April 5, 1954 



Cyclic Regeneration of Carbon Dioxide Acceptor 



1763 



Degradation of Ribulosb 



H 



Phenyl- 

 hydrazine 



HC=N— N— CH. 



C=N— N— CHi 

 I H 



HC— OH 



I 

 HC— OH 



i 

 CHjOH 



H 



HIO, 



NaHCO, 



HC=N— N— CH, 



i=N— N— CH, -f HCOOH 4- HCHO 

 I H 



CHO 



1, 2, 3 4 5 



CHjOH 



I 



c=o 



I 



HCOH 



I 

 HCOH 



I 

 CHjOH 



Ce(C10.)«- 

 H-^ 



COi 



2 



+ 4HCOOH 



PtOa 



HIO, 



CHjOH 



I 

 HCOH 



I 

 HCOH 



I 

 HCOH 



CHjOH 



ber by atmospheric pressure, the detachable face fell off 

 and the leaf was pulled into boiling ethanol. An estimated 

 exposure time of 0.4 sec. was obtained. The radioactive 

 products were extriicted and analyzed in the usual way. 

 In other experiments, longer exposure times were obtained 

 by holding the detachable face in position. 



Degradation of Sugars. — The reactions used for the degra- 

 dation of the radioactive ribulose and scdohcptulosc are 

 shown in the accompany flow sheets 



All radioactive material was purified on two-dimensional 

 paper chromatograms.'° Radioactive sedoheptulose was 

 converted to the anhydride by liealing at 11)0° with acid- 

 treated Dowex-.')0 for one hour, followed by chromatography 

 to separate the resulting equilibrium mixture. 



Formation of the Osazones. — The hexosc and hcptose 

 osazones were made in the usual manner with phenylhydra- 

 zine hydrochloride, sodium acetate and acetic acid. Usu- 

 ally about 25 mg. of sugar carrier was used for the reaction. 

 Sedoheptulose osazone cocrystallized with glucosazone 

 sufficiently well for fructose to be used as carrier with sedo- 

 heptulose activity. 



The radioactive arabinosazone was made by the method of 

 Haskins, Hann and Hudson" with 10 mg. of arabinose car 

 rier. The osazone was recrystallized once and diluted, as 

 desired for each degradation, with pure crystalline, non- 

 radioactive arabinosazone from a similar large-scale prepa- 

 ration . 



Oxidation of Osazones. — The recrystallized osazones were 

 treated with periodate in bicarbonate buffer as described by 

 Topper and Hastings.'^ The reaction mixture was frac- 

 tionated to obtain all the products by centrifuging and 

 thoroughly washing the raesoxaldehyde osazone; distilling 

 the supernate plus washings to dryness in vacuo and treating 

 the distillate with dimedon to obtain the formaldehyde de- 

 rivative; and acidifying and vacuum distilling the residue 

 to obtain the formic acid, which was counted as barium for- 

 mate. All products were recrystallized before counting. 



Cerate Oxidation of Ketoses. — The oxidation of the car- 

 bonyl carbon of a ketose to CO2 by cerate ion was performed 

 according to the method described by Smith." To a solu- 

 tion of an aliquot portion of radioactivity plus weighed 

 carrier (sedoheptulosan or fructose) was added a slight ex- 

 cess of 0.5 M cerate ion" in 6 iV perchloric acid, the final 

 concentration of acid being 4 N. The resultant COj was 



(11) W. T Haskins. R. N. Hano and C. S Hudson. This Jodrnal, 

 U, 1766 (1946). 



(12) Y. J. Topper and A B Hastings./. Biol C*«m, 1T9, 1255 (1949). 



(13) G Frederick Smith. "Cerate Oiidimetry." G Frederick Smith 

 Chemical Company. Columbua, Ohio, 1942. 



(14) We are indebted to Prof. John C. Speck, Jr , of Michigan State 

 College, East Lansing. Michigan, for valuable data and suggestions re- 

 garding the use of cerate in these oxidations. 



2HCH0 4- 3HCOOH 

 1,5 2.3,4 



swept with nitrogen into COj-free sodium hydroxide. The 

 reaction was allowed to proceed for one hour at room tem- 

 perature and then the COj was precipitated and counted as 

 barium carbonate. In all cases the theoretical amount of 

 carbon dioxide was evolved. 



Formation and Oxidation of Sugar Alcohols. — The radio- 

 active sugars were hydrogenated with platinum oxide as de- 

 scribed previously' and chromatographed on paper for puri- 

 fication. Carrier ribitol or voleraitol was added to an ali- 

 quot of radioactive alcohol and a slight excess of paraperiodic 

 acid was added. The reaction was allowed to stand at room 

 temperature for 6-7 hours. Then the formic acid and form- 

 aldehyde were distilled off in vacuo. After the formic acid 

 was titrated with barium hydroxide, the fonnaldehyde was 

 redistilled and precipitated as formyldimedon. Both the 

 residue of barium formate and the formyldimedon were re- 

 crystallized before plating and counting. 



Bacterial Oxidation of Hepitola from the Reduction of 

 Sedoheptulose. — The radioactive reduction products of 

 sedoheptulose gave only one spot on chromatography. 

 After elution these were oxidized by Acetobacter suboxydans 

 in a small-scale modification of the usual method." Two 

 mg. of volemitol and about 100 d- of solution of radioactive 

 heptitols were placed in a 7-mm. diameter vial. An amount 

 of yeast extract sufficient to make a 0.5% solution was 

 added. The vial was sterilized, then inoculated from a 24- 

 hour culture of Acetobacter and left for a week at room tem- 

 perature in a humid atmosphere. 



When the bacteria were centrifuged from the incubation 

 mixture and the supernatant solution was chromatographed, 

 three radioactive spots were obtained. The two major 

 spots were mannoheptulose and sedoheptulose, the oxidation 

 products of volemitol. The third had R, values very simi- 

 lar to those of fructose and cochromatographed with au- 

 thentic guloheptulose'" ( if f in phenol = 0.47; Ri in butanol- 

 propionic acid-water = 0.24). After treatment with Do- 

 wex-50 in the acid form at 100° for one hour, this third com- 

 pound gave a new compound which cochromatographed 

 vrith guloheptulosan (Ri in phenol = 0.62; i?i in butanol- 

 propionic acid-water = 0.30). It thus appeared that the 

 radioactive heptitols are volemitol and 0-sedoheptitol which 

 cochromatograph in the solvents used. 



Both mannoheptulose and guloheptulose have carbon 

 chains inverted from the original sedoheptulose. In the 

 small-scale fermentations, however, the oxidation appeared 

 to be incomplete. The original alcohol did not separate 

 chromatographically from mannoheptulose. Therefore, 



(16) (a) L. C. Stewart, N. K. Richtmyer and C. S. Hndsoo, TBM 

 JoDRNiL, 74, 2206 (1952); (b) we wish to express oor •ppreci»tion to 

 Dr. R. Clinton Fuller for his development of the micro-fermentation. 



(16) We wish to thank Dr. N. K. Richtmyer for his generons gift of 

 crystalline guloheptulosan. 



95 



